NF-κB通过Slc1a2调控树突状细胞成熟及功能的研究

Regulatory role of Slc1a2 on the maturation and function of dendritic cells

目的研究核因子-κB(NF-κB)通过溶质载体家族1成员2(Slc1a2)对树突状细胞(DC)成熟及功能的调控作用。方法采用Slc1a2特异性的siRNA以及过表达Slc1a2真核表达载体转染小鼠骨髓来源的DC,实时荧光定量RT-PCR和Western Blot检测敲降及过表达效率,采用流式细胞术检测DC表面分子CD40、CD80及主要组织相容性复合体Ⅱ(MHCⅡ)的表达和酶联免疫吸附测定(ELISA)法检测细胞因子白细胞介素(IL)-12、IL-6和转化生长因子-β(TGF-β)的分泌考察敲降Slc1a2对DC成熟和功能的影响,并考察过表达Slc1a2对DC成熟和功能的影响以及采用混合淋巴细胞培养检测Slc1a2对T细胞增殖能力的影响及ELISA检测IL-17A的分泌水平,通过流式细胞术分析DC的FITC相对荧光强度变化考察过表达Slc1a2对抗原吞噬的能力,最后采用NF-κB抑制剂甲苯酰苯丙氨酸氯甲基酮(TPCK)预处理DC,考察TPCK对DC内Slc1a2的表达及DC成熟的影响。结果脂多糖刺激成熟的DC中Slc1a2高表达(P<0.001)。在DC中敲降Slc1a2降低了DC成熟及刺激CD4^(+)T细胞增殖的能力(P<0.001),抑制了IL-17的分泌(P<0.01)。过表达Slc1a2增强了DC成熟及刺激CD4^(+)T细胞增殖的能力,促进IL-17A的分泌(均P<0.01)。NF-κB抑制剂TPCK预处理DC抑制了脂多糖诱导的Slc1a2在mRNA和蛋白水平的表达。结论NF-κB调控了DC内Slc1a2的表达,其表达水平影响了DC的成熟及功能。

Objective To investigate the regulatory effects of nuclear factor-κB(NF-κB)on dendritic cell(DC)maturation and function through solute carrier family 1 member 2(Slc1a2).Methods Mouse bone marrow-derived DCs were transfected with Slc1a2-specific siRNA and an overexpression Slc1a2 eukaryotic expression vector.The real-time fluorescence quantitation(RT-PCR)and Western Blot methods were used to detect knockdown and overexpression efficiency.The expression of surface molecules(CD40,CD80)and major histocompatibility complexⅡ(MHCⅡ)of DCs was detected by flow cytometry.ELISA was used to detect the secretion of the cytokines interleukin(IL)-12,IL-6,and transforming growth factor-β(TGF-β).The effects of knockdown of Slc1a2 on DC maturation and function and the effects of overexpression of Slc1a2 on DC maturation and function were reflected by the above assay results.A mixed lymphocyte culture assay was used to investigate the effect of Slc1a2 on T cell proliferation,and an ELISA was used to detect the lavel of IL-17A.Changes in the relative fluorescence intensity of FITC in DCs were analyzed by flow cytometry to investigate the ability of Slc1a2 overexpression on antigen phagocytosis.Finally,DCs were pretreated with an NF-κB inhibitor,toluoylphenylalanine chloromethyl ketone(TPCK),and the effect of TPCK on the expression of Slc1a2 in DCs and DC maturation was examined.Results Slc1a2 expression was found to be high in DC treated with lipopolysaccharides(LPS)(P<0.001).The knockdown of Slc1a2 decreased DC maturation and ability to stimulate the proliferation of CD4^(+)T cells(P<0.001)and inhibited IL-17 secretion(P<0.01).Overexpression of Slc1a2 promoted DC maturation and ability to stimulate the proliferation of CD4^(+)T cells(all P<0.01)Pretreatment of DC with the NF-κB inhibitor TPCK inhibited the expression of Slc1a2 at mRNA and protein levels induced by LPS.Conclusions NF-κB regulates Slc1a2 expression,which affects the maturation and function of DC.